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Appl Environ Microbiol, April 1998, p. 1276-1282, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Characterization of the rrnB Operon of the Plant Pathogen Rhodococcus fascians and Targeted Integrations of Exogenous Genes at rrn Loci

Agustín Pisabarro,1 António Correia,2 and Juan F. Martín1,*

Area of Microbiology, Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of León, 24071 León, Spain,1 and Departamento de Biologia, Universidad de Aveiro, 3800 Aveiro, Portugal2

Received 3 June 1997/Accepted 17 January 1998

A 6.0-kb SalI DNA fragment containing an entire rRNA operon (rrnB) was cloned from a cosmid gene bank of the phytopathogenic strain Rhodococcus fascians D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5' and 3' ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with the rrn operons of Bacillus subtilis and other gram-positive bacteria. Four copies of the rrn operons were identified by hybridization with an rrnB probe in R. fascians type strain ATCC 12974 and in the virulent strain R. fascians D188. However, another isolate, CECT 3001 (= NRRL B15096), also classified as R. fascians, produced five rrn-hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal to rrnB was constructed for targeted integration of exogenous genes at the rrn loci. Transformants carrying the exogenous chloramphenicol resistance gene (cmr) integrated in different rrn operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

* Corresponding author. Mailing address: Area of Microbiology, Faculty of Biology, University of León, 24071 León, Spain. Phone: (34-87) 291505. Fax: (34-87) 291506. E-mail: degjmm{at}unileon.es.




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